The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by radioactive materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over a substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity. In particular, the use of enzyme-triggered chemiluminescent 1,2-dioxetanes has achieved extremely high sensitivity for detection of enzyme-linked analytes.
Further increases in assay sensitivity or speed are needed to expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. One way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of reagents which generate light with a higher efficiency or for a greater length of time. This result may be obtained by using chemiluminescent substrates with a higher inherent chemiluminescence efficiency or by the use of enhancer substances which increase the efficiency of light emission.
In addition to increasing light emission, enhancer substances should ideally also be stable under conditions of use. It is desirable that the rate of the chemiluminescence reaction in the presence of the enhancer be relatively rapid as manifested by maximum light intensity and, in an enzyme-initiated reaction, the time required to reach maximal intensity. Further improvement in one or more of these characteristics over compounds known in the art would provide advantages in the application of chemiluminescence in analysis.